Method and apparatus for computer controlled rare cell, including fetal cell, based diagnosis

ABSTRACT

A computer controlled method for detecting and diagnosing a rare cell type in a tissue sample is provided, said method comprising treating the tissue sample such that it generates a first signal indicative of the presence at a location of a rare cell, detecting the first signal, treating the location at which the first signal is detected to generate a second signal indicative of a diagnostically useful cellular characteristic and detecting the second signal. The first signal can be morphological or a color present in a sought cell either before or after staining. The second signal can be generated by in situ PCR or PCR in situ hybridization. In one preferred embodiment, the rare cell type is a fetal cell in a maternal blood tissue sample, said sample consisting of a smear of unenriched maternal blood. In another embodiment, the method is used to diagnose or genotype cancer cells in a blood or tissue biopsy sample.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/091,360, filed on Mar. 4, 2002, which is a continuation of U.S.patent application Ser. No. 09/724,384 filed Nov. 28, 2000, which is adivisional of U.S. patent application Ser. No. 09/421,956 filed Oct. 20,1999, which is a continuation of PCT/US99/10026 filed May 7, 1999, whichclaims priority of U.S. Provisional Patent Application No. 60/084,893,filed May 9, 1998, all disclosures of which are incorporated byreference herein in their entirety.

1. FIELD OF THE INVENTION

The present invention relates to computer controlled methods andapparatus for obtaining and preparing cell samples and for identifying arare cell of interest from a field of cells and making a diagnosis basedon a characteristic of a rare cell selected in the field. In oneimportant embodiment, the invention relates to obtaining and preparing amaternal blood sample for fetal cell based prenatal diagnosis.

2. BACKGROUND OF THE INVENTION

The advent of DNA based prenatal diagnosis for human genetic disordershas led to the development of a number of new diagnostic methods. Thesediagnostic methods permit early detection and consequently informeddecisions and intervention with respect to fetus having a geneticdisorder. These methods, however, have a number of disadvantages. Eachof the new diagnostic methods with which this discussion is concernedrequires that a sample of isolated fetal cells be obtained, so that theDNA of the fetus may be examined or tested for signs of specific geneticdisorders. The disadvantages of these modern methods stem primarily fromthe need to obtain a sample of fetal cells. Currently, fetal cells areobtained by invasive procedures requiring obstetric intervention byamniocentesis or by chorionic villus sampling. These highly specializedprocedures carry a small, but significant, risk to the fetus. Early inpregnancy, the level of risk to the fetus is high and the number ofcells obtained is low. Therefore, results of these procedures often arenot obtained until 18-20 weeks of pregnancy.

One modern procedure for obtaining fetal cells relies on leakage offetal cells into the maternal circulation. By simply drawing a sample ofmaternal blood, it is theoretically possible to obtain fetal cellmaterial in a sufficient quantity for prenatal diagnosis by DNA basedmethods. Obtaining fetal cells from the maternal blood circulationavoids any risk to the fetus and can be undertaken as early as 10-12weeks of pregnancy.

Fetal cells which have been detected in the maternal blood circulationinclude trophoblasts, lymphocytes and nucleated erythrocytes.Trophoblasts were the first fetal cells to be identified in the maternalblood circulation, due to their large size. However, nucleatederythrocytes have generated the greatest degree of interest as sourcesof genetic material for prenatal diagnosis due to their rarity in theadult blood circulation, their abundance in fetal blood and theirlimited life span. These factors combine to reduce errors indistinguishing fetal cell material from maternal cell material. Fetalcells circulating in the maternal blood have a life span ranging from afew weeks (for the nucleated erythrocytes) to a few years (for thelymphocytes).

Although they are consistently present in the maternal bloodcirculation, fetal cells are very rare, severely limiting theirdiagnostic utility. Estimates of the concentration of fetal cells withinthe maternal blood circulation vary widely, from a high level of 1 fetalcell in 10⁵ maternal cells, to a low level of one fetal cell in 10⁹maternal cells. Thus, a 10 ml sample of maternal blood will ordinarilycontain between about 10 and 100 fetal cells. Throughout thisdescription, the concentration of fetal cells found in a freshly drawnmaternal blood sample, prior to any further treatment, is referred to asthe “naturally present concentration” of fetal cells, typically, but notnecessarily, within the above ranges. Also throughout this description,the term “unenriched maternal blood” shall refer to a sample of maternalblood which contains only a naturally present concentration of fetalcells.

Since the naturally present concentration of fetal cells in unenrichedmaternal blood is so low, in order to obtain a diagnosticallysignificant sample of fetal cells modern techniques include methods ofphysically isolating the fetal cells from the maternal cells in thesample. In essence, modern techniques are methods of concentrating thefetal cells within a sample, i.e., enriching the sample, for example byremoving excess maternal cells, without removing fetal cells. Thesemethods are extremely difficult to perform, often fail to isolate asufficient number of fetal cells to be diagnostically significant andsometimes fail to provide a sample of a sufficient number of undamagedfetal cells of adequate purity for reliable subsequent diagnosis.

3. SUMMARY OF THE INVENTION

It is desired to provide a computer controlled method and apparatus fordetecting and diagnosing a rare cell type in a tissue sample, saiddiagnosis being based upon a characteristic of that rare cell. It isfurther desired to provide a computer controlled method and apparatusfor detecting fetal cells in a blood preparation and performing a fetalcell based prenatal diagnosis that solves the above-identified problems,which overcomes such other problems and meets such other goals as willbe apparent to the person skilled in this art after reading adescription of the invention.

Generally, the invention provides a computer-implemented method ofprocessing body fluid or tissue sample image data, the method comprisingcreating a subset of a first image data set representing an image of abody fluid or tissue sample taken at a first magnification, the subsetrepresenting a candidate blob which may contain a rare cell creating asubset of a second image data set representing an image of the candidateblob taken at a second magnification, the subset of the second data setrepresenting the rare cell and storing the subset of the second data setin a computer memory.

In general, a subset of a first image data set can be created byobserving an optical field of a monolayer of cells from a body fluid ortissue sample using a computerized microscopic vision system to detect asignal indicative of the presence of a rare cell.

The method further comprises contacting a body fluid or tissue sample ata location corresponding to each candidate blob represented in thesubset of the first image data set, with a reagent to generate amedically significant signal. This method provides the advantage ofbeing able to remove from further processing a body fluid or tissuesample for which no subset of the first data set representing acandidate blob is created. The signal can be measured to determinewhether it is a significant signal level. The first and/or the secondimage data subsets can be transformed into a representation that is moresuitable for control and processing by a computer as described herein.In a preferred embodiment, the image data is transformed from an RGB(Red Green Blue) signal into an HLS (Hue Luminescence Saturation)signal. Filters and/or masks are utilized to distinguish those cellsthat meet pre-selected criteria and eliminate those that do not, andthus identify rare cells.

In another aspect of the invention, there is provided a method ofoperating a laboratory service, the method comprising steps of receivinga body fluid or tissue sample, creating a body fluid or tissue samplesmear, operating a computerized microscope so that a software programautomatically identifies a rare cell in the smear and detecting amedically significant signal in the rare cell.

In yet another aspect of the invention, there is provided computersoftware product including a computer-readable storage medium havingfixed therein a sequence of instructions which when executed by acomputer direct performance of steps of detecting and diagnosing a rarecell type. The cells encompass: creating a subset of a first image dataset representing an image of a body fluid or tissue sample taken at afirst magnification, the subset representing a candidate blob which maycontain a rare cell creating a subset of a second image data setrepresenting an image of the candidate blob taken at a secondmagnification, the subset of the second data set representing the rarecell and storing the subset of the second data set in a computer memory.

In general, a subset of a first image data set can be created asdescribed above. The steps further encompass contacting a body fluid ortissue sample at a location corresponding to each candidate blobrepresented in the subset of the first image data set, with a reagent togenerate a medically significant signal. This provides the advantage ofbeing able to remove from further processing a body fluid or tissuesample for which no subset of the first data set representing acandidate blob is created. There is an optional step by which the signalcan be measured to determine whether it is of a significant level.Another optional step encompasses transformation of one or both of thefirst and the second image data subsets into a representation that ismore suitable for control and processing by a computer as describedherein. In a preferred embodiment, the image data is transformed from anRGB (Red Green Blue) signal into an HLS (Hue Luminescence Saturation)signal. Filters and/or masks are utilized to distinguish those cellsthat meet pre-selected criteria and eliminate those that do not.

According to one aspect of the invention, there is provided a method ofpreparing a sample of cells for a diagnostic procedure. The sample ofcells is obtained and fixed as a monolayer on a substrate, the sample ofcells including a rare cell which is present in the sample at no greaterthan one in every 10,000 cells (i.e. no greater than 0.01%).kl Anoptical field covering at least a portion of the sample of cells isobserved using a computerized microscopic vision system for a signalindicative of the presence of a rare cell. The signal is detected, andcoordinates where the signal is detected are identified, for thediagnostic procedure. In one embodiment the rare cell is present at nogreater than 0.001% of the cells. In other embodiments the rare cell ispresent at no greater than 0.0001%, 0.00001% or even 0.000001%.

In one particularly important embodiment, the rare cell is a fetal cellin a sample of cells from maternal blood. In a preferred embodiment, thesample contains only a naturally present concentration of fetal cellswhich can be no greater than 0.001%, 0.0001%, 0.00001%, 0.000001% oreven 0.0000001%.

In another specific embodiment of the invention, the rare cell type tobe detected and diagnosed is a cancer cell found in a sample of cells ortissue from an animal or patient. The sample can be blood or other bodyfluid containing cells or a tissue biopsy. As an illustration of thisembodiment, cancer cell markers described in Section 5, infra, e.g. GM4protein, telomerase protein or nucleic acids, and p53 proteins ornucleic acids, may be used in the generation of the first or secondsignal, in a manner to be determined by the specific application of theinvention.

In one embodiment of the invention, when the rare cell type is presentin the sample, the method of the invention detects the rare cell type ata frequency of no less than 80%. In other embodiments, the detectionfrequencies are no less than 85%, 90%, 95% and 99%.

According to one particularly important embodiment of the invention,there is provided a method of preparing a sample of blood for adiagnostic procedure, which includes: preparing a smear of a sample ofunenriched maternal blood containing a naturally present concentrationof fetal cells; observing an optical field covering a portion of thesmear using a computerized microscopic vision system for a signalindicative of the presence of a fetal cell; detecting said signal; andidentifying, for the diagnostic procedure, coordinates within the smearat which the signal is detected.

In one embodiment, the signal is further processed to representmorphological measurements of the rare cell. In another embodiment, thecells are treated with a label to enhance the optical distinction ofrare cells from other cells. In this embodiment, the signal can be, forexample, from a label which selectively binds to the rare cells. Inanother embodiment, the diagnostic procedure involves moving to thecoordinates identified and magnifying the optical field until the imageis of an isolated rare cell.

In some embodiments, the optical field is stepped over a sequence ofportions of the cells covering substantially all of the cells. This maybe achieved, for example, by moving the cells on the substrate undercontrol of the computerized microscopic vision system relative to a lensof the computerized microscopic vision system. In another embodiment,the coordinates at which the first signal was obtained are identified,and then the rare cell at those coordinates specifically is contactedafter the coordinates have been identified.

According to another aspect of the invention, there is provided a methodof obtaining from a sample of cells a signal having diagnosticsignificance relative to a rare cell in the sample of cells. The rarecell is present in the sample at no greater than one in every 10,000cells. The method includes preparing a monolayer of the sample of cellsfixed on a substrate. The rare cell is contacted with an agent togenerate a diagnostic signal, the diagnostic signal having thediagnostic significance. The monolayer is observed using a computerizedmicroscopic vision system to obtain the diagnostic signal. In someembodiments, the diagnostic signal can be used to identify the rarecell. In other embodiments, a locating signal can be used to identifythe rare cell, and the diagnostic signal is obtained after the cell islocated.

In one embodiment, the rare cell is present in the sample at no greaterthan one in every 10,000 cells (i.e. no greater than 0.01% of thecells). In other embodiments, the rare cell is present at no greaterthan 0.001%, 0.00001% or even 0.000001%. In one particularly importantembodiment, the rare cell is a fetal cell in a sample of cells frommaternal blood. Preferably the sample contains only a naturally presentconcentration of fetal cells which can be no greater than 0.001%,0.0001%, 0.00001%, 0.000001% or even 0.0000001%.

According to an important embodiment of the invention, there is provideda method of obtaining from a sample of unenriched maternal blood,containing a naturally present concentration of fetal cells, a signalhaving diagnostic significance relative to the fetal cells. The methodincludes: preparing a smear of the sample of unenriched maternal blood;observing the smear using a computerized microscopic vision system toobtain a first signal indicative of the presence of a fetal cell;contacting the fetal cell with an agent to generate a second signal, thesecond signal having the diagnostic significance; and observing thefetal cell using the computerized microscopic vision system to obtainthe second signal. In one embodiment, the smear can comprise at least250 μl of the unenriched maternal blood and even can comprise at least500 μl of the unenriched blood.

As described above, the first signal can be further processed torepresent morphological measurements of the rare cell. Likewise, thecells can be treated with a label to enhance optical distinctions ofrare cells from other cells, such as maternal cells. To achieve this,the first signal can be from a label which selectively binds to the rarecell, such as a fetal cell. Likewise, as above, the step of observingcan involve stepping an optical field over a sequence of portions of thecells, which can be accomplished, for example, by moving the cells orthe substrate under control of the computerized microscopic visionsystem relative to a lens of the computerized microscopic vision system.In any of the foregoing embodiments, the cells can be prepared on asubstrate, and a coordinate system can be calibrated to the substrate sothat coordinates of the rare cell identified in one step can be returnedto later in another step. Likewise, the substrate in certain importantembodiments has a length that is 10 times its width, the substrate beingsubstantially elongated in one direction. The length can even be 20times the width. The substrate can be a flexible film, and in oneimportant embodiment, is an elongated flexible film that can carry arelatively large volume of cells, such as would be provided from arelatively large volume of smeared maternal blood.

In any of the foregoing embodiments, the first signal and the secondsignal can be selected whereby they do not mask one another when bothare present. Likewise, in any of the foregoing embodiments, the secondsignal can be generated by in situ PCR or PCR in situ or fluorescence insitu hybridization (FISH).

In one important embodiment, the substrate is a plurality of substrateson which the sample of cells is prepared, such as a plurality of smearsof maternal blood, each of the plurality including a total of at least 5μl of the sample. A rare cell-containing substrate (in which the firstsignal is obtained) is identified. Then, only the rare cell-containingsubstrate/substrates which has/have been identified is/are treated togenerate the second signal.

According to yet another aspect of the invention, there is provided amethod of performing a diagnosis for a fetus, using an unenriched sampleof maternal blood containing naturally present fetal cells. This methodincludes: preparing a smear of at least 250 μl of the sample ofunenriched maternal blood; identifying a fetal cell within the smear;contacting the fetal cell with an agent that produces a diagnosticsignal; and observing the diagnostic signal. In one importantembodiment, the step of identifying can comprise observing cells withinthe smear, using a computerized microscopic vision system, measuring asignal produced by the observed cells indicative of the presence offetal cells, and defining coordinates at which the measured signalindicates the presence of the fetal cell. Important embodiments directedto volumes of the maternal blood, substrate configurations and so forthare as described above.

According to yet another aspect of the invention, there is provided amethod of obtaining from a sample of unenriched maternal bloodcontaining a naturally present concentration of fetal cells, an image ofa substantially isolated fetal cell. This method includes: preparing asmear of at least 250 μl of the sample of unenriched maternal blood;observing the smear with a computerized microscopic vision system for asignal indicative of the presence of a fetal cell; identifyingcoordinates at which the signal is observed; moving to the coordinatesidentified, an optical field including an image of the fetal cell; andmagnifying the optical field until the image is of an isolated fetalcell. Important embodiments directed to volumes of the maternal bloodsample, substrate configurations and so forth are as described above.

According to yet another aspect of the invention, there is provided adevice for screening fetal cells contained within a smear of anunenriched sample of maternal blood containing a naturally presentconcentration of fetal cells, comprising: a flexible film having thereona smear of at least 250 μl of maternal blood. In one embodiment, theflexible film has thereon a smear of at least 500 μl of maternal blood.In one important embodiment, the flexible film is an elongated film, thelength being at least 10 times the width. It is particularly preferredthat the flexible film include marking coordinates, whereby thecomputerized microscopic vision system described herein can locate acell relative to a point on the film, permitting that cell to bereturned to at a later time, if desired.

According to yet another aspect of the invention, there is provided adevice for screening rare cells contained within a sample of cells at aconcentration of no greater than one rare cell for every 10,000 cells inthe sample of cells. The device is a flexible film having fixed thereonthe sample of cells, wherein the flexible film is at least five incheslong. In one preferred embodiment the flexible film has a length atleast 10 times its width. In another important embodiment, the flexiblefilm includes marking coordinates, whereby the computerized microscopicvision system described herein can locate a cell relative to a point onthe film, permitting the cell to be returned to at a later time, ifdesired.

According to another aspect of the invention, there is provided a devicefor dispensing materials to a specific location on a slide. The deviceincludes a microscopic vision system for detecting a signal indicativeof the presence of a rare cell in a sample of cells. The device alsoincludes means for identifying the coordinates of the rare cell in anoptical field. The device further has attached to it a dispenser fordispensing a volume of material and means for moving the dispenser tothe coordinates whereby the volume of material may be dispensed upon therare cell. The material dispensed can be reagents such as a label, PCR,primers, and the like.

According to another important embodiment of the invention, the need forscanning large areas of microscopic preparations in the minimum possibleamount of time is met by the use of an apparatus or system that providesa “composed” image. It is based on the simultaneous use of an array ofcomputer controlled objective lenses, arranged on a support system andhaving the capacity to focus on a microscopic preparation. Each of theobjective lenses is connected to a charge coupled device camera, hereinreferred to as a CCD camera, being connected to image acquisitionhardware installed in a host computer. The images are stored in thecomputer memory and they are combined in an appropriate side to sidefashion, so that a “composed” image is formed in the computer memory.The “composed” image can be further processed as a unity, using any kindof imaging procedures to detect specific features that are in question.The significant advantage of the described system consists in itscapacity to acquire images simultaneously from a number of objectivelenses, thus minimizing the time needed to process large areas of thesample in a manner that is inversely proportional to the number ofobjectives used.

The “composing” system can process any kind of microscopic preparationusing either transmitted or reflected light. It is particularly usefulwhere large numbers of samples need to be processed imposing significanttime constraints, for example, for processing large numbers ofmicroscopic biological preparations for screening and/or diagnosticpurposes, etc.

4. BRIEF DESCRIPTION OF THE DRAWINGS

In the accompanying drawings, in which like reference designationsindicate like elements:

FIG. 1 is a flow chart summarizing the method of one aspect of theinvention;

FIG. 2 is a block diagram of an analysis system used in one embodimentof one aspect of the invention;

FIG. 3 is a flow chart of stage I leading to detecting the first signal;

FIGS. 4A and 4B taken together are a flow chart of stage II leading todetecting the first signal;

FIG. 5 is a flow chart of detection of the second signal;

FIG. 6 is a schematic representation of a variation of an apparatusembodying aspects of the invention, using a continuous smear technique;

FIG. 7 is a block diagram of an analysis and reagent dispensing systemused in one embodiment of one aspect of the invention;

FIG. 8 is an outline of a multiple objective microscopy system; and

FIG. 9 is an image “composition” method.

5. DETAILED DESCRIPTION OF THE INVENTION

The invention will be better understood upon reading the followingdetailed description of the invention and of various exemplaryembodiments of the invention, in connection with the accompanyingdrawings. While the detailed description explains the invention withrespect to fetal cells as the rare cell type and blood as the body fluidor tissue sample, it will be clear to those skilled in the art that theinvention can be applied to and, in fact, encompasses diagnosis based onany rare cell type and any body fluid or tissue sample for which it ispossible to create a monolayer of cells on a substrate.

Body fluids and tissue samples that fall within the scope of theinvention include but are not limited to blood, tissue biopsies, spinalfluid, meningeal fluid, urine, alveolar fluid, etc. For those tissuesamples in which the cells do not naturally exist in a monolayer, thecells can be dissociated by standard techniques known to those skilledin the art. These techniques include but are not limited to trypsin,collagenase or dispase treatment of the tissue.

In an important specific embodiment, the invention is used to detect anddiagnose fetal cells. Our approach is directly opposite to that taken byothers seeking a non-invasive method for performing fetal cell basedprenatal diagnosis. Rather than attempting substantially to enrich theconcentration of fetal cells within a maternal blood sample, ourapproach involves identifying fetal cells within an unenriched maternalblood sample and subsequently performing diagnostic procedures on thefetal cells so identified, in situ.

A summary of this new approach, shown in the flow chart of FIG. 1, is asfollows:

Prepare one or more blood smears from a sample of unenriched maternalblood 101;

Screen the one or more blood smears until a predetermined number offetal cells (e.g., nucleated erythrocytes) have been identified andtheir coordinates defined 103; and

Process those smears or coordinates of a smear at which fetal cells havebeen identified, diagnosing the presence or absence of a particulargenetic feature in the fetal cells 105.

In this approach, two signals are defined, referred to hereinafter asthe first signal and the second signal. As used herein, “signal” shouldbe taken in its broadest sense, as a physical manifestation which can bedetected and identified, thus carrying information. One simple anduseful signal is the light emitted by a fluorescent dye selectivelybound to a structure of interest. That signal indicates the presence ofthe structure, which might be difficult to detect absent the fluorescentdye.

Screening 103 is based on the first signal. The first signal, which inthis exemplary embodiment indicates cell identity, may be generated by afluorescent dye bound to an antibody against the hemoglobin.epsilon.-chain, i.e., embryonal hemoglobin, for example.

Alternatively, for example, a metric of each cell's similarity to thecharacteristic morphology of nucleated erythrocytes, discerned usingcell recognition algorithms may serve as the first signal. In yetanother example, the first signal may be a measure of the presence ofthe characteristic color of fetal hemoglobin after staining with eosinand acid hematoxylin. It should now be evident that any detectableindicator of the presence of fetal cells may serve as the first signal,subject to certain constraints noted below.

Diagnosing 105 is based on the second signal. The second signal, whichin this exemplary embodiment indicates the presence of a particulargenetic characteristic being tested for, may be generated, for example,by in situ PCR-amplification or PCR in situ hybridization or FISH. Cellsthat emit both signals, i.e., the cell is a fetal cell and contains thegenetic characteristic being tested for, will be scored. Counts may bemaintained of the number and strengths of the first and second signalsdetected.

In one embodiment, a specific nucleic acid sequence is detected by FISH.In an exemplary embodiment, FISH comprises hybridizing the denaturedtest DNA of the rare cell type, e.g. a fetal cell, with a denatureddioxygenin (DIG)-labeled genomic probe. The samples containing the testDNA are washed and allowed to bind to an anti-DIG antibody coupled to afluorophore. Optionally, a second layer of fluorophore (e.g. FITC) isadded by incubation with fluorophore-conjugated anti-Fab antibodies. Ina preferred embodiment, FISH comprises hybridizing the denatured DNA ofthe rare cell with a fluorescently labeled probe comprising DNAsequence(s) homologous to a specific target DNA region(s) directlylabeled with a particular fluorophore.

Automated sample analysis will be performed by an apparatus and methodof distinguishing in an optical field objects of interest from otherobjects and background. An example of an automated system is disclosedin our U.S. Pat. No. 5,352,613, issued Oct. 4, 1994. Furthermore, oncean object has been identified, the color, i.e., the combination of thered, green, blue components for the pixels that comprise the object, orother parameters of interest relative to that object can be measured andstored.

Another example of an apparatus and method for automated sample analysisis presented, infra, in Section 6, Exemplary Embodiments, in particularSections 6.2.1, 6.2.2 and 6.3, and is illustrated in FIGS. 3-5.

In one embodiment of the invention, the system consists of an automaticmicroscopical sample inspection system having:

a sample storage and loading and unloading module

a sample transporting mechanism to and from an automated stage thatmoves the sample under a microscope objective lenses array

an array of CCD cameras

a processing unit having a host computer, multiple controllers tocontrol all mechanical parts of the microscopy system and

a high speed image processing unit where the CCD cameras are connected.

An innovative feature of this embodiment of a computer controlled systemis an array of two or more objective lenses having the same opticalcharacteristics, depicted in FIG. 8. The lenses are arranged in a rowand each of them has its own z-axis movement mechanism, so that they canbe individually focused (801). This system can be equipped with asuitable mechanism so that the multiple objective holder can beexchanged to suit the same variety of magnification needs that a commonsingle-lens microscope can cover. Usually the magnification range oflight microscope objectives extends from 1× to 100×.

Each objective is connected to its own CCD camera (803). The camerafield of view characteristics are such that it acquires the full area ofthe optical field as provided by the lens.

Each camera is connected to an image acquisition device (804). This isinstalled in a host computer. For each optical field acquired, thecomputer is recording its physical location on the microscopical sample.This is achieved through the use of a computer controlled x-y mechanicalstage (805). The image provided by the camera is digitized and stored inthe host computer memory. With the current system, each objective lenscan simultaneously provide an image to the computer, each of whichcomprises a certain portion of the sample area. The lenses should beappropriately corrected for chromatic aberrations so that the image hasstable qualitative characteristics all along its area.

The imaged areas will be in varying physical distance from each other.This distance is a function of the distance at which the lenses arearranged and depends on the physical dimensions of the lenses. It willalso depend on the lenses' characteristics, namely numerical apertureand magnification specifications, which affect the area of the opticalfield that can be acquired. Therefore, for lenses of varyingmagnification/numerical aperture, the physical location of the acquiredimage will also vary.

The computer will keep track of the features of the objectives-array inuse as well as the position of the motorized stage. The storedcharacteristics of each image can be used in fitting the image in itscorrect position in a virtual patchwork, i.e. “composed” image, in thecomputer memory as shown in FIG. 9.

For example, when starting the host computer moves the sample stage toan initial (x₁,y₁) position. Following the acquisition of the images atthis position, the stage moves to a new (x₂,y₂) position, in a side-wisemanner. Then a new set of images is acquired and also stored. As shownin FIG. 9 at a certain step 1, the image segments denoted “1” arecaptured and stored. In step 2, the segments “2” are stored. In step 3,the segments “3” are stored. The complete image is “composed” in thecomputer memory as the successive image segments are acquired.

The host computer system that is controlling the above configuration, isdriven by software system that controls all mechanical components of thesystem through suitable device drivers. The software also comprisesproperly designed image composition algorithms that compose thedigitized image in the computer memory and supply the composed image forprocessing to further algorithms. Through image decomposition, synthesisand image processing specific features particular to the specific sampleare detected.

In all automated sample analysis embodiments of the invention, if thegeneration of the first signal is measured first, indicating cellidentity, the one or more smears will be observed using an automatedoptical microscope to delineate coordinates of a desired number of fetalcells. Only those smears found to contain fetal cells need be treated togenerate the second signal, indicating the presence of the particulargenetic characteristic being tested for. The automated image analysisalgorithms will search for the presence of the second signal atpredetermined coordinates of fetal cells and also at predeterminedcoordinates of control maternal cells. This process could be reversed,whereby the genetic abnormality signal is observed first, and then thecell emitting that signal could be observed to determine whether it is afetal cell. It is even possible to observe both signals simultaneously,searching only for the simultaneous presence of two signals at a singleset of coordinates or even a single signal which results from theinteraction of two components (e.g. a quenching of a first signal by apartner ‘signal’, the first signal being for the cell type and thepartner ‘signal’ being for the genetic abnormality).

The requirements and constraints on the generation of the first andsecond signals are relatively simple. The materials and techniques usedto generate the first signal should not interfere adversely with thematerials and techniques used to generate the second signal (to anextent which compromises unacceptably the diagnosis), and visa versa.Nor should they damage or alter the cell characteristics sought to bemeasured to an extent that compromises unacceptably the diagnosis.Finally, any other desirable or required treatment of the cells shouldalso not interfere with the materials or techniques used to generate thefirst and second signals to an extent that compromises unacceptably thediagnosis. Within those limits, any suitable generators of the first andsecond signals may be used.

This exemplary embodiment of the invention may be characterized thus:(i) rather than attempting to enrich (or to further enrich if alreadypartially enriched) the concentration of fetal cells within the maternalblood, fetal cells within the unenriched maternal blood sample areidentified for further processing; and (ii) a suitable single celldetection method, such as in situ PCR and/or PCR in situ hybridizationis performed to determine the presence of a genetic characteristic beingtested for, in some instances only on smears or coordinates of smearsthat have already been stained and processed, and within which fetalcells have been detected.

Although in an important embodiment, the maternal blood used contains anaturally present concentration of fetal cells, the invention is meantto embrace also maternal blood which has been partially enriched forfetal cells. According to the prior art, the goal was to obtain as muchenrichment as possible, to achieve concentrations of fetal cells greaterthan one fetal cell per 1000 maternal cells. It in particular was thegoal to completely isolate fetal cells from maternal cells. According tothe invention, cell samples are used where the rare cell is present atno greater than one in every 10,000 cells (i.e. no greater than 0.01%).Thus, simple procedures may be employed to partially enrich the maternalblood sample for fetal cells, such as using simple fractionationprocedures (e.g. centrifugation or density gradients) and the like. Theprocedure falls within the scope of the invention when the sample ofcells containing the rare cell is used where the rare cell is present atno greater a concentration than 0.01%. As mentioned above, the inventionalso in very important embodiments is used where the concentration ofthe rare cell is 0.001%, 0.0001%, 0.00001%, 0.000001%, and even0.0000001%. The typical concentration of fetal cells in maternal bloodis between one fetal cell in 10⁵ maternal cells to one fetal cell in 10⁹maternal cells. Thus, the invention is useful over the full-range ofconcentrations of fetal cells in maternal blood as typically occursnaturally.

In one specific embodiment of the invention, when the rare cell type ispresent in the sample, the method of the invention detects the rare celltype at a frequency of no less than 80%. In other embodiments, thedetection frequencies are no less than 85%, 90%, 95% and 99%.

In addition to the detection of genetic abnormalities in a developingfetus, the above-described method is applicable to any situation whererare event detection is necessary. In particular, the invention can beapplied in any situation where a signal from a rare cell is to bedetected where the rare cell is present at a concentration no greaterthan one rare cell for every 10,000 other cells. The invention isparticularly applicable to those circumstances where the rare cell canbe distinguished phenotypically from the other cells whereby the rarecell first is identified using a first signal, and then the geneticcharacteristics of the cell identified are determined using a secondsignal.

Any chromosomal abnormality or Mendelian trait could be diagnosed usingthe present rare cell technology. The only prerequisite is knowledge ofthe underlying molecular defect. Use of single fluorophores for thetagging of an individual allele creates an upper limit as to the numberof mutations that can be tested simultaneously, however use ofcombinatorial chemistry increases enormously the number of allelespecific mutations that can be tagged and detected simultaneously.Chromosomal abnormalities that fall within the scope of the inventioninclude but are not limited to Trisomy 21, 18, 13 and sex chromosomeaberrations such as XXX, XXY, XYY. With the use of combinatorialchemistry, the methods of the invention can be used to diagnose amultitude of translocations observed in genetic disorders and cancer.Mendelian disorders that fall within the scope of the invention includebut are not limited to cystic fibrosis, hemochromatosis,hyperlipidemias, Marfan syndrome and other heritable disorders ofconnective tissue, hemoglobinopathies, Tay-Sachs syndrome or any othergenetic disorder for which the mutation is known. The use ofcombinatorial chemistry dyes allows for the simultaneous tagging anddetection of multiple alleles thus making it possible to establish theinheritance of predisposition of common disorders, e.g. asthma and/orthe presence of several molecular markers specific for cancers, e.g.,prostate, breast, colon, lung, leukemias, lymphomas, etc.

One particularly important use of the invention is in the field ofcancer. Cancer cells of particular types often can be recognizedmorphologically against the background of non-cancer cells. Themorphology of cancer cells therefore can be used as the first signal.Heat shock proteins also are markers expressed in most malignantcancers. Labeled antibodies, such as fluorescently-tagged antibodies,specific for heat shock proteins can be used to generate the firstsignal. Likewise, there are antigens that are specific for particularcancers or for particular tissues, such as Prostate Specific Antigen,and antibodies specific for cancer or tissue antigens, such as ProstateSpecific Antigen can be used to generate a first signal for such cancercells.

Once a cancer cell has been identified by the first signal, a secondsignal can be generated for providing more information about the cancer.For example, the lifetime risk of breast cancer approaches 80-90% inwomen with a germ line mutation in either BRCA1 or BRCA2. A variety ofmutations in these genes are known and have been reported.

Prostrate cancer is somewhat unique in its presentation to thepathologist of a bewildering array of histologies difficult to assign todiagnostic criteria. It is important to analyze and record the geneticalterations found in prostate cancer, with the objective of correlationto the pathology and natural history of the disease. Such geneticalterations include known alterations in P53, ras, Rb, cyclin-dependentkinases, oncogenes and tumor suppressors. T-cell receptor generearrangements are known in large granular lymphocyte proliferations.T-cell receptor delta gene rearrangements are known in acutelymphoblastic leukemia and non-Hodgkin's lymphoma.

Thus, rare cancer cells in a background of other cells can be identifiedand characterized according to the invention. The characterization mayinclude a confirmation of a diagnosis of the presence of the cancercell, a determination of the type of cancer, a determination of cancerrisk by determining the presence of a marker of a genetic change whichrelates to cancer risk, etc. Some of the following markers can be usedeither as the first or the second signals depending on the purpose towhich the invention is directed, as will be recognized by those ofordinary skill in the art. The markers include:

Human tumor specific antibody GM4. It preferentially reacts withmelanomas and neuroblastomas.

Bone morphogenic proteins (BMPs). Bone metastasis is a common event inprostate cancer and some of the BMPs are expressed in prostate cancercells.

Growth regulatory genes. Alterations in the structure and expression ofgrowth regulatory genes can lead to the initiation of malignanttransformation and tumor progression.

Protein tyrosine kinases. Such kinases are over-expressed in esophagealcancer and play an important role in regulation of proliferation.

Telomerase (hTRT). Elevated expression of hTRT occurs in some cancertissues.

p53, c-erbB-2 and p21ras. These genes are over expressed in ovarianneoplasms. Development of ovarian carcinoma is the end result of actionof several cancer causing genes.

BCL-2 family of proto-oncogenes. These genes are critical regulators ofapoptosis whose expression frequently becomes altered in human cancers(including some of the most common types of leukemias and lymphomas).

eKi-ras and c-myc. Mutation of these genes is implicated in tumorinitiation and progression in rectal cancer.

APC, p53 and DCC. These are implicated in colorectal tumorcarcinogenesis. Treatment strategies need to be coordinated withknowledge of the behavior of the tumor based on its genetic fingerprint.

Markers of genetic changes enable assessment of cancer risk. Theyprovide information on exposure to carcinogenic agents. They can detectearly changes caused by exposure to carcinogens and identify individualswith a particularly high risk of cancer development. Such markersinclude LOH on chromosome 9 in bladder cancer, and chromosome 1pdeletions and chromosome 7, 17 and 8 gains/losses detected in colorectaltumorigenesis.

Development of lung cancer requires multiple genetic changes. Activationof oncogenes includes K-ras and myc. Inactivation of tumor suppressorgenes includes Rb, p53 and CDKN2. Identification of specific genesundergoing alteration is useful for the early detection of cellsdestined to become malignant and permits identification of potentialtargets for drugs and gene-based therapy.

Mutations in genes that lie in the retinoblastoma pathway are implicatedin the pathogenesis of many tumor types. Two critical componentsinvolved in tumor progression are p16/CDKN2A and CDK4. Alterations inthe former is well documented in multiple cancers including melanoma.Alterations in the latter are rarer.

Mutations in one of four mismatch repair genes (hMSH2, hMLH1, HPMS1 andhPMS2) account for 70% of HNPCC.

Chromosome 11p15.5 is an important tumor suppressor gene region showingLOH in Wilms tumor, rhabdomyosarcoma, adrenocortical carcinoma and lung,ovarian and breast cancer.

Identification of numerically infrequent leukemic cells via uniquegenomic fusion sequences include MLL-AF4 and PML/RAR (in acutepromyelocytic leukemia).

T-cell receptor gene rearrangements are known in large granularlymphocyte proliferations.

T-cell receptor delta gene rearrangements are known in acutelymphoblastic leukemias and non-Hodgkin's lymphoma.

FAP is caused by mutations in the APC gene resulting in multipleadenomas of the colorectal mucosa.

The invention is described in connection with observing “monolayers” ofcells. Monolayer has a specific meaning as used herein. It does notrequire confluence and can involve single cell suspensions. It meanssimply that the cells are arranged whereby they are not stacked on topof one another, although all of the cells can be separated from oneanother. Thus, monolayers can be smears of single cell suspensions orcan be a thin layer of tissue. Any solid or exfoliative cytologytechnique can be employed.

The invention also has been described in connection with identifying apair of signals, one which identifies a target rare cell such as a fetalcell and another which is useful in evaluating the state of the cellsuch as a fetal cell having a genetic defect. It should be understoodthat according to certain embodiments, only a single signal need bedetected. For example, where a fetal cell carries a Y chromosome and thediagnosis is for an abnormality on the Y chromosome, then the signalwhich identifies the genetic abnormality can be the same as that whichidentifies the fetal cell. As another example, a single signal can beemployed in circumstances where the observed trait is a recessive trait.A pair of signals also can be used to detect the presence of two allelesor the existence of a condition which is diagnosed by the presence oftwo or more mutations in different genes. In these circumstances thepair of signals (or even several signals) can identify both thephenotype and the cell having that phenotype. Such embodiments will beapparent to those of ordinary skill in the art.

6. EXEMPLARY EMBODIMENTS

6.1. Smear Preparation

Smears were prepared from 10 μl aliquots of whole blood on glassmicroscope slides. Smears were prepared from both cord blood andmaternal circulating blood and allowed to air dry.

6.1.1. Cell Fixation.

Fixation of smears prior to cell permeabilization for in situ PCR or PCRin situ hybridization was under one of three conditions. (i) Smears werefixed in ice-cold methanol for 10 minutes-16 hours. (ii) Smears werefixed in ice-cold 10% buffered formalin for 10 minutes-16 hours. (iii)Smears were fixed in 2% paraformaldehyde for 10 minutes-16 hours.Following fixation, smears were washed three times in phosphate bufferedsaline, at room temperature, for 10 minutes. Smears were then air-dried.

6.1.2. Cell Staining

Polychrome Staining:

The smears were covered with Wright's stain and incubated for one to twominutes at room temperature. Distilled water (2.5 ml) was then added todilute the stain and incubation at room temperature continued for 3-6minutes. The stain was then washed off rapidly with running water and a1:10 dilution of Giemsa stain added to the slide. Incubation was at roomtemperature for 5 minutes and the stain was then washed off rapidly withrunning water. The smears were then air-dried.

Antibody Staining:

The smears were covered with anti-embryonal hemoglobin (hemoglobin.epsilon.-chain) monoclonal antibody and incubated at room temperaturefor one to three hours. The slides were then washed twice in phosphatebuffered saline, at room temperature, for 5 minutes. Secondary antibody(anti-mouse antibody conjugated to phycoerythrin) was then added and theslide incubated at 37° C. for 30 minutes. The slides were then washedtwice in phosphate buffered saline, at room temperature, for 5 minutesand air-dried.

Fetal Hemoglobin Staining:

Smears were fixed in 80% ethanol for 5 to 10 minutes, then rinsed withtap water and air dried. Acid citrate-phosphate buffer (37.7 ml 0.1Mcitric acid, 12.3 ml 0.2M Na₂HPO₄, pH 3.3) was pre-warmed in a coplinjar in a 37° C. water bath. The fixed smears were then added to thecoplin jar and incubated at 37° C. for 5 minutes. The smears were thenrinsed with tap water and stained with 0.1% hematoxylin for one minute.The smears were then rinsed with tap water and stained with 0.1% eosinfor one minute. The smears then underwent a final rinse in tap water andwere air-dried.

Cell Permeabilization:

Cell permeabilization was attained by incubation in either proteinase K(1-5 mg/ml in phosphate buffered saline) or pepsin (2-5 mg/ml in 0.01Mhydrochloric acid). Incubation was at room temperature for 1-30 minutes.Following permeabilization, smears were washed in phosphate bufferedsaline, at room temperature, for 5 minutes, then in 100% ethanol, atroom temperature, for one minute. Smears were then air-dried.

PCR In Situ Hybridization:

For PCR in situ hybridization, smears were overlaid with 50 μlamplification solution. Amplification solution comprised 10 mM Tris-HCl,pH 8.3, 90 mM potassium chloride, 1-5 mM magnesium chloride, 200 μMdATP, 200 μM dCTP, 200 μM dGTP, 200 μM dTTP, 1 μM forward primer, 1 μMreverse primer and 5-10 units thermostable DNA polymerase in aqueoussealing reagent. A glass coverslip was then lowered onto theamplification solution and the slide transferred to a thermal cycler.Following an initial denaturation step at 94° C. for 4 minutes, theslide was then subjected to 25-35 cycles of amplification, where eachcycle consisted of denaturation at 94° C. for one minute, annealing at55° C. for one minute and extension at 72° C. for one minute. Thecoverslip was then removed by incubation of the slide in phosphatebuffered saline for 10 minutes at room temperature, and the slideair-dried. Fluorescein labeled oligonucleotide probe in hybridizationbuffer (600 mM sodium chloride, 60 mM sodium citrate, 5% dextransulfate, 50% formamide) was then added and the slide covered with aglass cover slip, and incubated at 94° C. for 10 minutes then at 37° C.for one hour. The coverslip was then removed by incubation of the slidein phosphate buffered saline for 10 minutes at room temperature and theslide then washed twice for 5 minutes in phosphate buffered saline atroom temperature. The smear was then covered with protein block solution(1% bovine serum, 2.5% goat serum, 0.2% Tween-20) and incubated at roomtemperature for 10 minutes. The solution was then removed and the slidewashed three times in phosphate buffered saline for 5 minutes at roomtemperature. The smear was then covered with mouse anti-fluoresceinmonoclonal antibody and incubated at room temperature for 20 minutes.The solution was then removed and the slide washed three times inphosphate buffered saline for 5 minutes at room temperature. The smearwas then covered with biotinylated goat anti-mouse F(ab)₂ and incubatedat room temperature for 20 minutes. The solution was hen removed and theslide washed three times in phosphate buffered saline for 5 minutes atroom temperature. The smear was then covered with alkaline phosphataseconjugated streptavidin and incubated at room temperature for 20minutes. The solution was then removed and the slide washed twice inphosphate buffered saline for 5 minutes at room temperature. Alkalinephosphatase substrate solution (50 mg/ml BCIP, 75 mg/ml NBT) was thenadded to the smear and the slide incubated at 37° C. for 10 minutes-twohours. The slide was then washed twice in distilled water at roomtemperature for 5 minutes and air-dried.

In Situ PCR:

For in situ PCR, smears were overlaid with 50 l amplification solution.Amplification solution comprised 10 mM Tris-HCl, pH 8.3. 90 mM potassiumchloride, 15 mM magnesium chloride, 200 μM dATP, 200 μM dCTP, 200 .mu.MdGTP, 0.5 μM [R110]dUTP, 1 μM forward primer, 1 μM reverse primer and5-10 units thermostable DNA polymerase in aqueous sealing reagent. Aglass coverslip was then lowered onto the amplification solution and theslide transferred to a thermal cycler. Following an initial denaturationstep at 94° C. for 4 minutes, the slide was then subjected to 25-35cycles of amplification, where each cycle consisted of denaturation at94°. C. for one minute, annealing at 55° C. for one minute and extensionat 72° C. for one minute. The coverslip was then removed by incubationof the slide in phosphate buffered saline for 10 minutes at roomtemperature and the slide air-dried.

6.2. Automated Smear Analysis

Automated smear analysis has been briefly summarized, above. Theapparatus and method used in the exemplary embodiment is now described.

6.2.1. Apparatus

The block diagram of FIG. 2 shows the basic elements of a systemsuitable for embodying this aspect of the invention. The basic elementsof the system include an X-Y stage 201, a mercury light source 203, afluorescence microscope 205 equipped with a motorized objective lensturret (nosepiece) 207, a color CCD camera 209, a personal computer (PC)system 211, and one or two monitors 213, 215.

The individual elements of the system can be custom built or purchasedoff-the-shelf as standard components. Each element will now be describedin somewhat greater detail.

The X-Y stage 201 can be any motorized positional stage suitable for usewith the selected microscope 205. Preferably, the X-Y stage 201 can be amotorized stage that can be connected to a personal computer andelectronically controlled using specifically compiled software commands.When using such an electronically controlled X-Y stage 201, a stagecontroller circuit card plugged into an expansion bus of the PC 211connects the stage 201 to the PC 211. The stage 201 should also becapable of being driven manually. Electronically controlled stages suchas described here are produced by microscope manufacturers, for exampleincluding Olympus (Tokyo, Japan), as well as other manufacturers, suchas LUDL (NY, USA).

The microscope 205 can be any fluorescence microscope equipped with areflected light fluorescence illuminator 203 and a motorized objectivelens turret 207 with a 20× and an oil immersion 60× or 63× objectivelens, providing a maximum magnification of 600×. The motorized nosepiece207 is preferably connected to the PC 211 and electronically switchedbetween successive magnifications using specifically compiled softwarecommands. When using such an electronically controlled motorizednosepiece 207, a nosepiece controller circuit card plugged into anexpansion bus of the PC 211 connects the stage 201 to the PC 211. Themicroscope 205 and stage 201 are set up to include a mercury lightsource 203, capable of providing consistent and substantially evenillumination of the complete optical field.

The microscope 205 produces an image viewed by the camera 209. Thecamera 209 can be any color 3-chip CCD camera or other camera connectedto provide an electronic output and providing high sensitivity andresolution. The output of the camera 209 is fed to a frame grabber andimage processor circuit board installed in the PC 211. A camera found tobe suitable is the SONY 930 (SONY, Japan).

Various frame grabber systems can be used in connection with the presentinvention. The frame grabber can be, for example a combination of theMATROX IM-CLD (color image capture module) and the MATROX IM-640 (imageprocessing module) set of boards, available from MATROX (Montreal,CANADA). The MATROX IM-640 module features on-board hardware supportedimage processing capabilities. These capabilities compliment thecapabilities of the MATROX IMAGING LIBRARY (MIL) software package. Thus,it provides extremely fast execution of the MIL based softwarealgorithms. The MATROX boards support display to a dedicated SVGAmonitor. The dedicated monitor is provided in addition to the monitorusually used with the PC system 211. Any monitor SVGA monitor suitablefor use with the MATROX image processing boards can be used. Onededicated monitor usable in connection with the invention is a ViewSonic4E (Walnut Creek, Calif.) SVGA monitor.

In order to have sufficient processing and storage capabilitiesavailable, the PC 211 can be any INTEL PENTIUM-based PC having at least32 MB RAM and at least 2 GB of hard disk drive storage space. The PC 211preferably further includes a monitor. Other than the specific featuresdescribed herein, the PC 211 is conventional, and can include keyboard,printer or other desired peripheral devices not shown.

6.2.2. Method

The PC 211 executes a smear analysis software program compiled inMICROSOFT C++ using the MATROX IMAGING LIBRARY (MIL). MIL is a softwarelibrary of functions, including those which control the operation of theframe grabber 211 and which process images captured by the frame grabber211 for subsequent storage in PC 211 as disk files. MIL comprises anumber of specialized image processing routines particularly suitablefor performing such image processing tasks as filtering, objectselection and various measurement functions. The smear analysis softwareprogram runs as a WINDOWS 95 application. The program prompts andmeasurement results are shown on the computer monitor 213, while theimages acquired through the imaging hardware 211 are displayed on thededicated imaging monitor 215.

In order to process microscopic images using the smear analysis program,the system is first calibrated. Calibration compensates for day to dayvariation in performance as well as variations from one microscope,camera, etc., to another. During this phase a calibration image isviewed and the following calibration parameters are set:

the color response of the system;

the dimensions or bounds of the area on a on a slide containing a smearto be scanned for fetal cells;

the actual dimensions of the optical field when using magnifications20.times. and 60× (or 63×); and

the minimum and maximum fetal nuclear area when using magnifications20.times. and 60× (or 63×).

6.2.3. Detection of the First (Identification) Signal

The fetal cell detection algorithm operates in two stages. The first isa pre-scan stage I, illustrated in the flow chart of FIG. 3, wherepossible fetal cell positions are identified using a low magnificationand high speed. The 20× objective is selected and the search of fetalcells can start:

The program moves the automated stage (FIG. 2, 201) to a preset startingpoint, for example one of the corners of a slide containing a smear(Step 301).

The x-y position of the stage at the preset starting point is recorded(Step 303) optical field.

The optical field is acquired (Step 305) using the CCD camera 209 andtransferred to the PC 211 as an RGB (Red/Green/Blue) image.

The RGB image is transformed (Step 307) to the ILLS(Hue/Luminance/Saturation) representation.

The Hue component is binary quantized (Step 309) as a black and whiteimage so that pixels with Hue values ranging between 190 and 255 are setto 0 (black) representing interesting areas (blobs), while every otherpixel value is set to 255 (white, background). The blobs representpossible fetal cell nuclear areas.

The area of each blob in the binary quantized image is measured. If, at20.times. magnification, it is outside a range of about 20 to 200 pixelsin size, the blob's pixels are set to value 255 (background); they areexcluded from further processing (Steps 311, 313, 315 and 317).

Then the coordinates of each blob's center of gravity (CG) arecalculated (Step 319), using a custom MATROX function. The center ofgravity of a blob is that point at which a cut-out from a thin, uniformdensity sheet of material of the blob shape would balance. Thesecoordinates are stored in a database along with the z-y position of thecurrent optical field, so the blob can be located again at the nextprocessing stage using higher magnification.

Additional optical fields are processed similarly, recording the x-yposition of each succeeding optical field, until the complete slide areis covered (Steps 321 and 323).

Stage II, illustrated in the flow chart of FIGS. 4A and 4B, includes thefinal fetal cell recognition process:

63.times. magnification is selected (Step 401).

The program moves the automated stage (FIG. 2, 201) so that thecoordinates of the first position of a CG found earlier, which ispossible fetal cell nuclear area, is at the center of the optical field(Step 403).

The optical field is acquired using the CCD camera (FIG. 2, 209) andtransferred to the computer as an RGB image (Step 405).

The RGB image is transformed to the HLS model (Step 407).

The program then generates a Luminance histogram (Step 409) by countingthe number of pixels whose Luminance value equals each possible value ofLuminance. The counts are stored as an array of length 256 containingthe count of pixels having a grey-level value corresponding to eachindex into the array.

The program next analyzes the Luminance distribution curve (Step 411),as represented by the values stored in the array, and locates the lastpeak. It has been found that this peak includes pixel values thatrepresent plasma area in the image. The function that analyzes theLuminance distribution curve: calculates a 9-point moving average tosmooth the curve; calculates the tangents of lines defined by points 10grey-level values distant; calculates the slopes of these lines indegrees;

finds the successive points where the curve has zero slope and setsthese points (grey-levels) as −1 if they represent a minimum (valley inthe curve) or 1 if they represent a maximum (peak in the curve); thenfinds the locations of peaks or valleys in the curve by finding theposition of a 1 or a −1 in the array of grey-level values.

The program then sets as a cut-off value the grey-level value of pixelslying in the valley of the Luminance distribution which occurs beforethe last peak of the distribution (Step 413).

Using this cut-off value, the program then produces (Step 415) a secondbinary quantized image. This is a black-and-white image in which pixelscorresponding to pixels in the Luminance image having grey-level valueslower than the cut off point are set to 255 (white) and pixelscorresponding to pixels in the Luminance image having grey-level valueshigher than the cut off point are set to 0 (black). The white blobs ofthis image are treated as cells while the black areas are treated asnon-cellular area.

A closing filter is applied (Step 417) to the second binary quantizedimage; in this way holes, i.e., black dots within white regions, areclosed.

The program now measures the area of the cells. If the area of any ofthe cells is less than 200 pixels then these cells are excluded, i.e.the pixels consisting these cells are set to pixel value 255 (black)(Step 419).

A hole fill function, found in the MIL, is applied to the remainingblobs (Step 412). The resulting binary quantized image, afterprocessing, is a mask whose white regions denote only cells.

Red blood cells are now distinguished from white blood cells based onthe Saturation component of the HLS image. The mask is used to limitprocessing to only the cell areas.

The program now counts the number of pixels whose Saturation value iseach possible value of Saturation. The counts are stored as an array oflength 256 containing the count of pixels having a grey-level valuecorresponding to each index into the array (Step 423).

The program now analyzes (Step 425) the Saturation distribution curve,as represented by the values stored in the array, and locates the firstpeak. This peak includes pixel values that represent areas contained inwhite blood cells.

The grey-level value that coincides with the first minimum (valley)after the peak is set as a cut-off point (Step 427).

Using this cut-off value the program produces (Step 429) a third binaryquantized image. Pixels corresponding to pixels in the Saturation imagehaving grey-level values higher than the cut-off point are set to 255(white). They constitute red blood cell areas. Pixels corresponding topixels in the Saturation image having grey-level values lower than thecutoff point are set to 0 (black). The white blobs of this third binaryquantized image are seeds for areas that belong to red blood cells.

A closing filter is applied (Step 431) to the third binary quantizedimage; in this way holes, i.e., black dots within white regions, areclosed.

A hole fill function, found in the MIL, is applied (Step 433) to theremaining blobs. The resulting binary quantized image, after processing,is a new mask that contains only white blood cells.

An erase border blob function of MIL is now applied (Step 435) to theremaining blobs, removing those which include pixels coincident with aborder of the image area. Such blobs cannot be included in furtherprocessing as it is not known how much of the cell is missing when it iscoincident with a border to the image area.

An erosion filter is applied 6 times to this mask; thus any connectedblobs (white blood cell seeds) are disconnected (Step 437).

A “thick” filter is applied 14 times (Step 439). The “thick” filter isequivalent to a dilation filter. That is, it increases the size of ablob by successively adding a row of pixels at the periphery of theblob. If a growing blob meets an adjacent blob growing next to it, thethick filter does not connect the two growing blobs. Thus adjacent blobscan be separated.

The first binary quantized mask (containing all the cells) and the thirdbinary quantized mask (containing the separated seeds of white bloodcells) are combined with a RECONSTRUCT_FROM_SEED MIL operator. A fourthmask thus constructed contains blobs (cells) copied from the first maskthat are allowed by the third mask and therefore represent white bloodcells (Step 441).

The blobs in the fourth mask are measured for their area andcompactness: Area (A) is the number of pixels in a blob; Compactness isderived from the perimeter (p) and area (A) of a blob, it is equal to:p2/4(A). The more convoluted the shape, the bigger the value. A circlehas the minimum compactness value (1.0). Perimeter is the total lengthof edges in a blob, with an allowance made for the staircase effectwhich is produced when diagonal edges are digitized (inside corners arecounted as 1.414, rather than 2.0). Blobs are retained in the fourthmask only if their area is between 1000 and 8000 pixels and they have acompactness less than 3, thus allowing for cells with relatively roughoutline. Blobs that touch the border of the image are excluded fromfurther processing (Step 443).

The fourth mask is applied to the Hue component in the following manner(Steps 445, 447, 449 and 451):

Pixels from the Hue component are copied to a new image retaining theirHue value, provided that their coordinates coincide with white (255)pixels in the “mask”; all other pixels in the new image are set to 0(black) (Step 445).

The pixel values in each of the contiguous non-0 pixel areas, i.e.,those blobs corresponding to images of red cells, are checked for valuesbetween 190 and 255. The number of such pixels in each blob is counted(Step 447).

If there are more than 200 such pixels, the blob represents a nucleatedred blood cell. The coordinates of the center of gravity of each suchcell are stored. The mask is binary quantized so that all pixels havingnon-0 values are set to 255 (white); and the mask is stored as aseparate Tagged Image File Format (TIFF) file (Step 449).

The program moves to the next stored coordinates for a possible fetalcell which do not coincide with any of the coordinates stored during theprevious step. The entire process is repeated until a preset number ofnucleated red blood cells have been identified. The results, includingthe nucleated red blood cell coordinates and the names of the respectivemask files, along with various characteristic codes for the blood slideare stored in a result text file. The nucleated red blood cells whosecoordinates are stored are the fetal cells sought (Step 451).

After fetal cells are identified, the second signal is generated, forexample by in situ CR or PCR in situ hybridization or FISH, as describedabove.

6.2.4. Detection of the Second Signal

A smear including in situ PCR or PCR in situ hybridization treated cellsis positioned on the stage (FIG. 2, 201). If necessary calibration stepsare taken, as before. Calibration permits the software to compensate forday to day variation in performance as well as variations from onemicroscope, camera, etc. to another. Detection of the second signal thenproceeds, as shown in the flow chart of FIG. 5, as follows:

Magnification objective 60× (63×) is chosen (Step 501).

The x-y stage is moved to the first fetal cell position according todata from the result file compiled from detection of the first signal,as described above (Step 503).

The optical field is acquired using the CCD camera (FIG. 2, 209) andtransferred to the computer (FIG. 2, 211) as an RGB image (Step 505).

The RGB image is transformed to the HLS model (Step 507).

The TIFF file containing the black and white mask is loaded as aseparate image (Step 509).

The pixels of the Hue component not corresponding to white areas in themask arc set to 0 (black) (Step 511).

The remaining areas, which represent fetal cells, are searched for pixelvalues corresponding to a signal produced following PCR. For example,the signal may be a color which arises due to the presence of alkalinephosphatase, i.e., red. The non black areas of the Hue component aresearched for pixel values ranging from 0 to 30 (Step 513).

The stage is moved to the next non-processed fetal cell and the aboveprocess is repeated (Step 515).

6.3. Variations

A number of variations on the above-described system and method are alsocontemplated and are encompassed by the present invention. Some of theseare now described. This description will also suggest others to thoseskilled in the art.

Each unenriched blood sample may be used to prepare smears on each of aplurality of individual microscope slides. When prepared in this way,each slide can undergo detection of the first signal. However, onlythose slides which the first signal is detected need be furtherprocessed to generate the second signal, and subsequently are analyzedto detect the second signal. Processing in this way permits the use ofconventional sample and slide-handling equipment.

In a variation illustrated schematically in FIG. 6, the unenriched bloodsample 601 is used to prepare a single, long smear on a flexiblesubstrate 603. The substrate 603 can have a length 10 or more times itswidth. For example, a strip of cellulose acetate film base with sprocketholes on either side could be used as the substrate. The strip carryingthe smear undergoes the processing steps described above in a continuousprocessing system, as shown in FIG. 6. After locations of fetal cellsare determined by detection of the first signal, segments of the smearincluding those locations are cut out of the continuous strip forgeneration and detection of the second signal.

In an alternative processing method using a single, long smear on aflexible substrate, the strip is divided into a plurality of individualsegments similar to microscope slides, before generating and detectingthe first signal. Processing proceeds as for individual microscopeslides.

The above variations, and similar variations, are advantageous in thatthe entire smear need not be processed for generation and detection ofthe second signal. Only those slides or segments in which the firstsignal is detected need undergo the further processing to generate anddetect the second signal.

In one aspect of the invention, a device is provided for dispensingreagents only to those portions of the smear where a rare cell isdetected. Referring to FIG. 7, an apparatus of the invention is shownincluding a reagent dispenser system. The reagent dispenser system canbe located for dispensing reagents to precise locations on the stage.This is particularly suited for dispensing reagents only of thecoordinates identified by a first signal, such as the coordinates of arare cell (e.g., a fetal cell and a maternal blood smear). The systemincludes a reagent dispenser 701 which is a housing for one or moremicropipettes located within the housing. The reagent dispenser isattached in this embodiment to the microscope and is positioned relativeto the stage in fixed relation to the microscope. The narrow tip of thereagent dispenser 701 is adjacent the stage 201. The opposite end of thereagent dispenser 701 has communicating therewith feedline 703 which isa tube or a housing carrying a plurality of tubes for deliveringreagents to the reagent dispenser 701. The feedline 703 is attachedremote from the reagent dispenser 701 to a first reagent container 705and a second reagent container 707. In the embodiment shown, thefeedline 703 is a housing through which passes feedline 703′communicating with reagent container 705 and feedline 703′ communicatingwith reagent container 707. A pump 709 is attached to feedline 703″ forpumping reagent from the reagent container 707 to the reagent dispenser701, and out the narrow tip of the reagent dispenser 701 onto the stageat a desired location. Another pump 709′ is attached to feedline 703′for delivering reagents from reagent container 705 to the reagentdispenser 701. The pumps are electronically controlled by PC211 usingspecifically compiled software commands indicated by “reagent control”.The reagents can be any one of the reagents described above inconnection with generating a signal.

In the embodiment shown, the reagent dispenser is attached to themicroscope. The reagent dispenser need not be attached to the microscopeand, instead, can be otherwise attached to any frame relative to the X-Ystage. The stage is shown as moving with respect to the reagentdispenser for locating the narrow tip of the reagent dispenser at aprecise location with respect to a slide on the stage. The slide on thestage can be moved to a different location, and the reagent dispensercan be itself moveably controlled to locate it relative to a set ofcoordinates in the slide. What is important is that, in an automatedfashion, the coordinates of a detected rare cell can be positioned withrespect to the dispensing end of the reagent dispenser, wherebymaterials may be delivered to a discrete location at the coordinates ofthe rare cell. If the reagent dispenser is controlled by a motor andmoveable with respect to a stage or a slide upon a stage, then thereagent dispenser can be provided with a sensor for locating itsposition with respect to the slide or stage. Thus, the slide on a stagecan be processed in series, with the microscope first locating thecoordinates on the slide of the rare cell. The slide then is next movedto a second processing area where the reagent dispenser is positioned atthe previously-identified coordinates in the slide and reagents aredelivered to generate the second signal. Optionally the slide could bemoved to a third station, such as a thermocycling station and then backto the microscope field for viewing.

It should be evident that different treatments of the smear are possiblewhen it is desired to identify a different cell type or to diagnose adifferent cellular characteristic. The biochemistry, morphologicalparameters and colors described above may each be varied in known waysto meet other diagnostic needs.

Computer and image processing technologies are constantly changing.Newer technologies which meet the needs of the above-described methodsand apparatus, while not specifically described here, are clearlycontemplated as within the invention. For example, certain conventionalpixel and image file formats are mentioned above, but others may also beused. Image files may be compressed using JPEG or GIF techniques nowknown in the art or other techniques yet to be developed. Processing maybe performed in an RGB color description space instead of the HLS spacecurrently used. Other color spaces may also be used, as desired by theskilled artisan, particularly when detection of a sought-aftercharacteristic is enhanced thereby.

While the embodiments of the invention have been described in connectionwith unenriched samples of maternal blood, aspects of the invention maybe practiced on conventionally enriched or partially enriched maternalblood samples, as well. The use of a computer-controlled microscopicvision system to identify and to diagnose fetal cells within the sampleis applicable to samples covering a full range of fetal cellconcentrations. As has been discussed above, the use of such a system isparticularly advantageous when used in connection with unenrichedmaternal blood samples.

The present invention has now been described in connection with a numberof particular embodiments thereof. Additional variations should now beevident to those skilled in the art, and are contemplated as fallingwithin the scope of the invention, which is limited only by the claimsappended hereto and equivalents thereof.

1. An automated diagnostic method for detecting abnormalities associatedwith a rare cell using an computerized automated microscopy systemoperatively configured to automatically search a microscope slide, saidmethod comprising the steps of: treating said cell sample with adiagnostic label selective to a diagnostic condition that may bedetected in said rare cell(s); affixing a portion of said cell samplecontaining said rare cell(s) as a monolayer on a microscope slide;automatically detecting by said computerized automated microscopy systemthe position of said rare cell based upon detection of pre-determinedparameters associated with said rare cell(s); automatically detecting bysaid computerized automated microscopy system whether a signalindicative of said diagnostic label is associated with said detectedposition of said rare cell(s); and automatically providing a putativediagnosis based on detection of said diagnostic label being associatedwith the position of one or more detected rare cell(s).
 2. The method ofclaim 1, wherein the rare cell is present at no greater than 0.001% ofcells in the sample of cells.
 3. The method of claim 2, wherein the rarecell is present at no greater than 0.0001% of cells in the sample ofcells.
 4. The method of claim 2, wherein the rare cell is present at nogreater than 0.00001% of cells in the sample of cells.
 5. The method ofclaim 1, wherein the computerized automated microscopy system used hastwo or more objectives.
 6. An automated diagnostic method for detectingabnormalities associated with a rare cell, found in a cell sampleapplied to a microscope slide at a concentration no greater than onecell in 10,000 cells, using an computerized automated microscopy systemoperatively configured to automatically search a microscope slide, todetect signals from said slide, and to process said signals, said methodcomprising the steps of: treating said cell sample with a selectiveidentification label of said rare cell to label said rare cell with asignal detectable by said automated microscopy system; treating saidcell sample with a diagnostic label selective to a diagnostic conditionthat may be detected in said rare cell, said diagnostic label producinga signal which is detectable by said automated microscopy system;affixing a portion of said cell sample containing said rare cell(s) as amonolayer on a microscope slide such that the monolayer comprises nogreater than one cell in 10,000 cells; detecting by said automatedmicroscopy system a signal indicative of said identification labelassociated with said rare cell and from said signal detectedautomatically determining the position of said rare cell(s) on saidslide; detecting by said automated microscopy system a signal indicativeof said diagnostic label associated with the position of said rarecell(s); automatically providing a putative diagnosis based on detectionof said diagnostic label being associated with one or more detected rarecell(s).
 7. The method of claim 6, wherein the rare cell is present atno greater than 0.001% of cells in the sample of cells.
 8. The method ofclaim 7, wherein the rare cell is present at no greater than 0.0001% ofcells in the sample of cells.
 9. The method of claim 8, wherein the rarecell is present at no greater than 0.00001% of cells in the sample ofcells.
 10. The method of claim 6, wherein the selective identificationsignal is processed to represent morphological measurements of the rarecell.
 11. The method of claim 6, further comprising the step of:treating said rare cell(s) labeled with said identification label withanother selective identification label to enhance optical distinction ofrare cell(s) from other cell(s).
 12. The method of claim 6, wherein thecomputerized automated microscopy system used in the method has two ormore objectives.
 13. A diagnostic method for detecting an abnormalityassociated with a rare cell, comprising: providing a computerizedmicroscope; forming on a substrate a monolayer of an unenriched sampleincluding the rare cell; using the computerized microscope to search thesubstrate for a first signal indicative of the rare cell; using thecomputerized microscope to detect a second signal indicative of the rarecell; and using the microscope to provide a diagnosis of the abnormalityof the rare cell based on the second signal.
 14. A diagnostic method fordetecting an abnormality associated with a rare cell using acomputerized microscope, comprising: forming on a substrate a monolayerof an unenriched sample including the rare cell; searching the substratefor a first signal indicative of the rare cell; positioning themicroscope relative to the first signal to detect a second signalindicative of the rare cell; and providing a diagnosis of theabnormality of the rare cell based on the second signal.
 15. A method ofobtaining from a sample of cells a signal having diagnostic significancerelative to a rare cell in the sample of cells, comprising the steps of:obtaining a monolayer of the sample of cells fixed on a substrate,wherein one or more rare cell(s) of interest is present in the monolayersample at no greater than one in every 10,000 cells; contacting the rarecell(s) of interest with an agent to generate a diagnostic signalassociated with one or more components of each of said rare cell ofinterest, the diagnostic signal having diagnostic significance;automatically determining the position of one or more said rare cell(s)of interest on said slide using a computerized microscopic visionsystem; automatically determining whether a diagnostic signal isassociated with the determined position of each of said rare cells ofinterest using said computerized microscopic vision system.
 16. Themethod of claim 15, wherein the rare cell is present at no greater than0.001% of cells in the sample of cells.
 17. The method of claim 16,wherein the rare cell is present at no greater than 0.0001% of cells inthe sample of cells.
 18. The method of claim 17, wherein the rare cellis present at no greater than 0.00001% of cells in the sample of cells.19. The method of claim 15, further comprising the step of: contactingsaid sample with an agent to generate a locating signal with respect tothe rare cell(s) of interest.
 20. The method of claim 19, furthercomprising the step of: determining the location of said rare cell(s) byway of said locating signal.
 21. The method of claim 19, furthercomprising the step of: further processing the locating signal torepresent morphological measurements of the rare cell(s).
 22. The methodof claim 15 wherein the sample of cells are derived from maternal bloodand the rare cell(s) is a fetal cell.
 23. A method of obtaining from asample of unenriched maternal blood containing a naturally presentconcentration of fetal cells, a signal having diagnostic significancerelative to the fetal cells, the method comprising: preparing a smear ofthe sample of unenriched maternal blood on a substrate; observing thesmear using a computerized microscopic vision system operativelyconfigured to read said substrate and to obtain a first signalindicative of the presence of a fetal cell; contacting the fetal cellwith an agent to generate a second signal, the second signal having thediagnostic significance; observing the fetal cell using the computerizedmicroscopic vision system to obtain the second signal; and countingoccurrences of the second signal in a plurality of fetal cells elicitingsaid first signal.
 24. The method of claim 23, wherein the first signalis further processed to represent morphological measurements of thefetal cells.
 25. The method of claim 23, wherein the first signal andthe second signal do not mask one another when both are present.
 26. Themethod of claim 23, further comprising the step of: calibrating acoordinate system to said substrate so that coordinates of said fetalcell(s) identified at one point in time can be returned to at a laterpoint of time.
 27. The method of claim 23, wherein the computerizedmicroscopic vision system has two or more objectives.